Targeted modification of mammalian genomes

Sorrell, David A.; Kolb, Andreas

doi:10.1016/j.biotechadv.2005.03.003

Cited by 91 publications

(77 citation statements)

References 161 publications

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“…Additionally, off-target recombination (2, 6) can result in deletion, chromosomal translocation, cytotoxicity, oncogenesis, and other adverse consequences that have all been documented (3-6, 32). Substantial off-target activity has also been observed with other classes of DNA-modifying proteins (1,33,34). Additional experiments are necessary to determine whether RecZFs can also cause these abnormalities, although the high level of specificity of plasmid integration suggests that this new class of enzymes will be less susceptible to these limitations.…”

Section: Discussionmentioning

confidence: 99%

“…The chief drawback of this approach is its relative inefficiency: HR is orders of magnitude less frequent than random plasmid integration (1). Several catalysts have been developed that dramatically enhance the efficiency of HR, including triplex forming oligonucleotides and adeno-associated viral vectors (1). A very promising method relies on endonucleases (e.g., homing nucleases such as I-SceI and zinc finger-nuclease fusion proteins) that recognize rare sites (23,29,38).…”

Section: Discussionmentioning

confidence: 99%

“…New tools are needed to accurately rewrite the genomic script and specifically alter genes, gene expression, and epigenetic state at any desired loci. To date, no enzyme-natural or synthetic-has been able to accurately modify only a single targeted site within the human genome (1). Scientists in biology, biotechnology, stem cell research, and gene therapy currently rely on naturally occurring enzymes to perform functions like DNA integration and excision.…”

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confidence: 99%

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Synthesis of programmable integrases

Gordley

Gersbach

Barbas

2009

Proc. Natl. Acad. Sci. U.S.A.

104

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Accurate modification of the 3 billion-base-pair human genome requires tools with exceptional sequence specificity. Here, we describe a general strategy for the design of enzymes that target a single site within the genome. We generated chimeric zinc finger recombinases with cooperative DNA-binding and catalytic specificities that integrate transgenes with >98% accuracy into the human genome. These modular recombinases can be reprogrammed: New combinations of zinc finger domains and serine recombinase catalytic domains generate novel enzymes with distinct substrate sequence specificities. Because of their accuracy and versatility, the recombinases/integrases reported in this work are suitable for a wide variety of applications in biological research, medicine, and biotechnology where accurate delivery of DNA is desired.recombinases ͉ zinc finger ͉ gene delivery ͉ gene targeting ͉ protein engineering T he postgenomic era of medicine will be defined by our ability to achieve biological control through genetic reprogramming. New tools are needed to accurately rewrite the genomic script and specifically alter genes, gene expression, and epigenetic state at any desired loci. To date, no enzyme-natural or synthetic-has been able to accurately modify only a single targeted site within the human genome (1). Scientists in biology, biotechnology, stem cell research, and gene therapy currently rely on naturally occurring enzymes to perform functions like DNA integration and excision. However, these enzymes recognize multiple sites within the human genome, often resulting in off-target DNA integration and chromosomal translocation (2-6). Our recent work with serine resolvases and invertases led us to hypothesize that we could use a modular approach that capitalizes on cooperative specificity to design synthetic enzymes that would uniquely recognize a single site within the 3 billion-base-pair human genome and allow us to deliver DNA specifically to this site (Fig. 1A) (7).In their native contexts, serine resolvases and invertases selectively recombine target sites within the same DNA molecule. This intramolecular specificity is assured by obligate assembly of large protein complexes, wherein accessory factors bound at neighboring sites impose topological and spatial constraints on the recombination reaction (8). Hyperactive mutants of several serine resolvases and invertases have been discovered that efficiently catalyze unrestricted recombination between minimal dimer-binding sites (Table S1) (9, 10). Furthermore, unlike other site-specific recombinases, serine resolvases and invertases are well suited to synthetic reengineering. These enzymes are modular in both structure and function, each comprised of a distinct catalytic domain flexibly tethered to a small helix-turnhelix DNA-binding domain (DBD). However, these DBDs are poorly suited for accurate genomic recombination (11) because the recognition motifs are short (4-6 bp) and degenerate (12, 13).In contrast, zinc finger DNA-binding proteins recognize target sites of v...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Synthesis of programmable integrases

Gordley

Gersbach

Barbas

2009

Proc. Natl. Acad. Sci. U.S.A.

104

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“…Alternatively the uC31 recombinase will only insert incoming sequence in a unidirectional manner due to the heterologous nature of its recognition sequences (known as att sites) (Thorpe and Smith 1998). A further potential for this particular integrase is the existence of att-like sites in the genomes of both human and mouse, facilitating the insertion of transgenic sequences without first having to introduce a recombinase-recognition sequence (Sorrell 2005). There are also likely to be similar sites in the hamster, though this has not yet been investigated to our knowledge.…”

Section: Recombinasesmentioning

confidence: 99%

“…Several recent reviews have addressed these advances in the context of gene therapy in particular as well as for basic research (Coates et al 2005;Vasquez et al 2001;Sorrell 2005). In this review, we will focus on the application of these tools in cell lines relevant to the biopharmaceutical industry.…”

Section: Introductionmentioning

confidence: 99%

Targeted genetic modification of cell lines for recombinant protein production

2007

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Considerable increases in productivity have been achieved in biopharmaceutical production processes over the last two decades. Much of this has been a result of improvements in media formulation and process development. Though advances have been made in cell line development, there remains considerable opportunity for improvement in this area. The wealth of transcriptional and proteomic data being generated currently hold the promise of specific molecular interventions to improve the performance of production cell lines in the bioreactor. Achieving this-particularly for multi-gene modification-will require specific, targeted and controlled genetic manipulation of these cells. This review considers some of the current and potential future techniques that might be employed to realise this goal.

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Site‐specific modification of the bovine genome using Cre recombinase‐mediated gene targeting.

Graham

Cole

Laible

2009

Biotechnology Journal

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Cre recombinase (Cre)-mediated targeted insertion of a transgene is a powerful technique that can be used to tailor genomes. When combined with somatic cell nuclear transfer it could offer an efficient way to generate transgenic livestock with site-specific genetic modifications that are free of antibiotic selection markers. We have engineered primary bovine fibroblasts to contain a chromosomal acceptor site with incompatible loxP/lox2272 sites for Cre-mediated cassette exchange and show for the first time that Cre-mediated targeting can be applied in these acceptor cells. Molecular characterization of the resulting cell clones revealed Cre-mediated transgene insertion efficiencies of up to 98% when antibiotic selection was used to identify transgene containing cell clones. Most clonal lines also contained random insertions of the targeting and Cre expression plasmids with only about 10% of the clones being exclusively modified by the intended targeted insertion. This targeting efficiency was sufficient to enable the isolation of correctly targeted clones without the help of antibiotic selection. Therefore, this recombinase-mediated insertion strategy has the potential to produce transgenic cattle from antibiotic selection marker-free somatic cells with transgenes inserted into proven genomic loci ensuring reliable expression levels.

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Targeted modification of mammalian genomes

Cited by 91 publications

References 161 publications

Synthesis of programmable integrases

Synthesis of programmable integrases

Targeted genetic modification of cell lines for recombinant protein production

Site‐specific modification of the bovine genome using Cre recombinase‐mediated gene targeting.

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